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Cited by 112Autophagy in Disease and TherapyGenetic Neurodegenerative DiseasesCell death mechanisms and regulation

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Autophagy activation and enhanced mitophagy characterize the Purkinje cells of pcd mice prior to neuronal death

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Chakrabarti, Lisa, Eng, Jeremiah, Ivanov, Nishi, Garden, Gwenn A, La Spada, Albert R·2009·Molecular Brain
DOI 10.1186/1756-6606-2-24PMC PMC2729476
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Abstract

Purkinje cells are a class of specialized neurons in the cerebellum, and are among the most metabolically active of all neurons, as they receive immense synaptic stimulation, and provide the only efferent output from the cerebellum. Degeneration of Purkinje cells is a common feature of inherited ataxias in humans and mice. To understand Purkinje neuron degeneration, investigators have turned to naturally occurring Purkinje cell degeneration phenotypes in mice to identify key regulatory proteins and cellular pathways. The Purkinje cell degeneration (pcd) mouse is a recessive mutant characterized by complete and dramatic post-natal, cell autonomous Purkinje neuron degeneration and death. As the basis of Purkinje cell death in pcd is unresolved, and contradictory data has emerged for the role of autophagy in Purkinje cell degeneration, we studied the mechanism of Purkinje cell death in pcd mice. BAX null status did not suppress Purkinje neuron death in pcd mice, indicating that classic apoptosis is not responsible for Purkinje cell loss. Interestingly, LC3 Western blot analysis and GFP-LC3 immunostaining of degenerating pcd cerebellum revealed activation of the autophagy pathway. Ultrastructural studies confirmed increased autophagy pathway activity in Purkinje cells, and yielded evidence for mitophagy, in agreement with LC3 immunoblotting of cerebellar fractions. As p62 levels were decreased in pcd cerebellum, our findings suggest that pcd Purkinje cell neurons can execute effective autophagy. However, our results support a role for dysregulated autophagy activation in pcd, and suggest that increased or aberrant mitophagy contributes to the Purkinje cell degeneration in pcd mice.

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Figure 1 Purkinje cell degeneration in pcd mice is characterized by dendrite retraction and shrinkage. (A-D) Toluidine blue staining of cerebellar sections. At 14 days of age, Purkinje cells of normal size and morphology (red arrows) are apparent in both wild-type control littermate mice (A) and in pcd5J homozygous mice (B). (C) At 21 days of age, Purkinje cells (red arrows) remain regularly spaced, retain normal size and morphology, and exhibit uniformly oriented dendritic processes (yellow arrowheads) in wild-type control mice. (D) However, by 21 days of age, dramatic histopathology becomes obvious in pcd5J homozygous mice, as significant drop-out of Purkinje cells has begun to occur. Many of the remaining Purkinje cells have aberrantly small and misshapen soma (red arrows), and display shortened and tortuous dendrites (yellow arrowheads). GCL = granule cell layer; PCL = Purkinje cell layer; ML = molecular layer. Scale bar corresponds to 20 μM.
Figure 1 Purkinje cell degeneration in pcd mice is characterized by dendrite retraction and shrinkage. (A-D) Toluidine blue staining of cerebellar sections. At 14 days of age, Purkinje cells of normal size and morphology (red arrows) are apparent in both wild-type control littermate mice (A) and in pcd5J homozygous mice (B). (C) At 21 days of age, Purkinje cells (red arrows) remain regularly spaced, retain normal size and morphology, and exhibit uniformly oriented dendritic processes (yellow arrowheads) in wild-type control mice. (D) However, by 21 days of age, dramatic histopathology becomes obvious in pcd5J homozygous mice, as significant drop-out of Purkinje cells has begun to occur. Many of the remaining Purkinje cells have aberrantly small and misshapen soma (red arrows), and display shortened and tortuous dendrites (yellow arrowheads). GCL = granule cell layer; PCL = Purkinje cell layer; ML = molecular layer. Scale bar corresponds to 20 μM.CC BY · PubMed Central
Figure 2 Purkinje cell degeneration in pcd mice isaccompanied by activation of the autophagy pathway. (A-C) Fluorescence microscopy of cerebellar sections from GFP-LC3 transgenic mice crossed with pcd5J homozygous mice. (A) At 28 days of age, wild-type mice transgenic for GFP-LC3 display a diffuse pattern of GFP staining. (B) GFP-LC3 - pcd5J homozygous mice, however, exhibit GFP puncta (arrows) in the cell bodies of their Purkinje cell neurons at 28 days of age. (C) These GFP-LC3 puncta are especially obvious at higher magnification, and can be seen in dendritic arbors as well as the cell body (arrows). GFP-LC3 = green; DAPI = blue. Scale bars correspond to 20 μM. (D) Western blot analysis of cerebellar protein lysates subjected to subcellular fractionation was also performed on protein samples from 28 day-old mice to assess autophagy activation. Both mitochondrial and cytosolic fractions from pcd5J homozygous mice showed a pronounced LC3-I to LC3-II shift. This increased LC3-II/LC3- I ratio is consistent with marked activation of the autophagy pathway in pcd5J homozygous mice. Note that visualized LC3 bands represent endogenous LC3 isoforms. Equivalent loading and the success of the fractionation were confirmed by re-probing the immunoblot with Cox-IV and SOD1 antibodies (not shown).
Figure 2 Purkinje cell degeneration in pcd mice isaccompanied by activation of the autophagy pathway. (A-C) Fluorescence microscopy of cerebellar sections from GFP-LC3 transgenic mice crossed with pcd5J homozygous mice. (A) At 28 days of age, wild-type mice transgenic for GFP-LC3 display a diffuse pattern of GFP staining. (B) GFP-LC3 - pcd5J homozygous mice, however, exhibit GFP puncta (arrows) in the cell bodies of their Purkinje cell neurons at 28 days of age. (C) These GFP-LC3 puncta are especially obvious at higher magnification, and can be seen in dendritic arbors as well as the cell body (arrows). GFP-LC3 = green; DAPI = blue. Scale bars correspond to 20 μM. (D) Western blot analysis of cerebellar protein lysates subjected to subcellular fractionation was also performed on protein samples from 28 day-old mice to assess autophagy activation. Both mitochondrial and cytosolic fractions from pcd5J homozygous mice showed a pronounced LC3-I to LC3-II shift. This increased LC3-II/LC3- I ratio is consistent with marked activation of the autophagy pathway in pcd5J homozygous mice. Note that visualized LC3 bands represent endogenous LC3 isoforms. Equivalent loading and the success of the fractionation were confirmed by re-probing the immunoblot with Cox-IV and SOD1 antibodies (not shown).CC BY · PubMed Central
Figure 3 Ultrastructural analysis of pcd mice reveals altered mitochondrial morphology, endoplasmic reticulum histopathology, and autophagosome engulfment of mitochondria in Purkinje cell soma. (A-C) We performed an extensive ultrastructural comparison of Purkinje cell soma between 18 day-old wild-type and pcd5J homozygous mice. Representative micrographs are shown here. (A) Control micrographs from a wild-type Purkinje cell soma. (B) Purkinje cell somae in pcd5J homozygous develop huge mitochondria that are likely to represent fusion at early stages (filled black arrows) and very swollen endoplasmic reticulum that is largely denuded of ribosomes (white arrows). Autophagic vacuoles appear and engulf mitochondria and other organelles (line arrows). We noted a significant number of Purkinje cells from pcd5J homozygous mice undergoing dark cell degeneration which was accompanied by increased numbers of autophagic vacuoles. Dark cell degeneration was uncommon in wild-type control micrographs (not shown). (C) and (D) are higher magnification views of inset regions in (B). Autophagic vacuoles containing electron dense cytoplasmic elements (arrows) are present, including vacuoles containing mitochondria (asterisks). Scale bars represent 1 μM.
Figure 3 Ultrastructural analysis of pcd mice reveals altered mitochondrial morphology, endoplasmic reticulum histopathology, and autophagosome engulfment of mitochondria in Purkinje cell soma. (A-C) We performed an extensive ultrastructural comparison of Purkinje cell soma between 18 day-old wild-type and pcd5J homozygous mice. Representative micrographs are shown here. (A) Control micrographs from a wild-type Purkinje cell soma. (B) Purkinje cell somae in pcd5J homozygous develop huge mitochondria that are likely to represent fusion at early stages (filled black arrows) and very swollen endoplasmic reticulum that is largely denuded of ribosomes (white arrows). Autophagic vacuoles appear and engulf mitochondria and other organelles (line arrows). We noted a significant number of Purkinje cells from pcd5J homozygous mice undergoing dark cell degeneration which was accompanied by increased numbers of autophagic vacuoles. Dark cell degeneration was uncommon in wild-type control micrographs (not shown). (C) and (D) are higher magnification views of inset regions in (B). Autophagic vacuoles containing electron dense cytoplasmic elements (arrows) are present, including vacuoles containing mitochondria (asterisks). Scale bars represent 1 μM.CC BY · PubMed Central
Figure 4 Ultrastructural analysis of pcd mice indicates accumulation of autophagic vacuoles and autophagosomes in Purkinje cell dendrites and axons. (A-B) We performed an extensive comparison of Purkinje cell dendrites between 18 day-old wild-type and pcd5J homozygous mice. Representative micrographs are shown here. (A) Dendrite process from a Purkinje cell neuron of a wild-type control. (B) In pcd5J homozygous mice, we noted frequent double-membrane bound structures in Purkinje cell dendrites (lined arrows), sometimes containing mitochondria (asterisks). (C) When we examined the Purkinje cell axons projecting to the deep cerebellar nuclei in pcd5J homozygous mice, we detected axons containing multiple autophagosome-like structures (arrows), containing mitochondria (asterisks) and other electron dense cellular components. Scale bars represent 1 μM.
Figure 4 Ultrastructural analysis of pcd mice indicates accumulation of autophagic vacuoles and autophagosomes in Purkinje cell dendrites and axons. (A-B) We performed an extensive comparison of Purkinje cell dendrites between 18 day-old wild-type and pcd5J homozygous mice. Representative micrographs are shown here. (A) Dendrite process from a Purkinje cell neuron of a wild-type control. (B) In pcd5J homozygous mice, we noted frequent double-membrane bound structures in Purkinje cell dendrites (lined arrows), sometimes containing mitochondria (asterisks). (C) When we examined the Purkinje cell axons projecting to the deep cerebellar nuclei in pcd5J homozygous mice, we detected axons containing multiple autophagosome-like structures (arrows), containing mitochondria (asterisks) and other electron dense cellular components. Scale bars represent 1 μM.CC BY · PubMed Central
Figure 5 p62 levels are decreased in pcd mice. Western blot analysis of p62 in cerebellar lysates from 27 day-old wild-type littermate controls (WT) and pcd5J homozygous mice (pcd) revealed decreased levels of p62 in pcd. Densitometric ratios of p62 to the tubulin loading control are shown at right.
Figure 5 p62 levels are decreased in pcd mice. Western blot analysis of p62 in cerebellar lysates from 27 day-old wild-type littermate controls (WT) and pcd5J homozygous mice (pcd) revealed decreased levels of p62 in pcd. Densitometric ratios of p62 to the tubulin loading control are shown at right.CC BY · PubMed Central

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Metadata

Authors
Chakrabarti, L., Eng, J., Ivanov, N., Garden, G. A., & La Spada, A. R.
Affiliations
University of Washington (US)
ORCID
Venue
Molecular Brain
Publisher
Springer Science and Business Media LLC
Published
2009-07-29
Volume
2
Issue
1
Pages
24-24
ISSN
1756-6606 (ISSN-L)
DOI
10.1186/1756-6606-2-24
PMC
PMC2729476
OpenAlex
W2028695079
Cited by
112
References
36
Citations by year
2026: 2 · 2025: 4 · 2024: 7 · 2023: 5 · 2022: 1 · 2021: 4 · 2020: 8 · 2019: 5 · 2018: 7 · 2017: 1 · 2016: 15 · 2015: 5 · 2014: 13 · 2013: 13 · 2012: 10
FWCI
4.95
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Molecular Brain (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health (EY-01730, HD02274)
  • Intellectual and Developmental Disabilities Research Center
  • National Eye Institute (EY 18106, EY-01730, R01EY014997, R21EY018106, P30EY001730, EY 14997)
  • National Institute of Neurological Disorders and Stroke (R01NS052535)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (P30HD02274, P30HD002274)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Genetic Neurodegenerative Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
Purkinje cells, Purkinje cell degeneration, autophagy, mitophagy, neuronal cell death, Bax, apoptosis, LC3, p62, cerebellum, hereditary ataxia, neurodegeneration
MeSH terms
Animals, Ataxia, Autophagy, Dendrites, Endoplasmic Reticulum, Mice, Neurologic Mutants, Mitochondria, Nerve Degeneration, Phagosomes, Phenotype, Purkinje Cells, bcl-2-Associated X Protein, Mice

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